Abstract Recently, we have revealed a new developmental pathway for CD4 T cells that is mediated by MHC class II expressing thymocytes. This finding provided an answer for several unexplainable observations of CD4 T cell development in humans. Human thymocytes express MHC class II and can mediate CD4 T cell selection and therefore two CD4 T cell populations are likely present in humans but not in mice. We named thymocyte-selected CD4 cells T-CD4 (Thymocyte-selected) and the other E-CD4 (Epithelial cell-selected) to reflect their selection pathway. Having established the new developmental pathway for CD4 T cells, we have begun investigating the function of T- CD4 T cells. Our preliminary data demonstrate that CD4 T cells possess a different cytokine production potential depending on their selection pathway. Unlike E-CD4 T cells, T-CD4 T cells can produce T helper (Th) 1 and 2 cytokines immediately after activation. Further examinations of T-CD4 T cells revealed that they make IL-4 in addition to IFN- even after being skewed to Th1 cells. This effector phenotype is acquired in the thymus and, remarkably, independent of Stat6. Interestingly, these characteristics are also found in NKT cells that are also selected on thymocytes. However, T-CD4 T cells are distinct from NKT cells since T-CD4 T cells require MHC class II-peptide complexes to develop, do not express NK1.1, and have a diverse TCR repertoire. Our new findings add another level of complexity in T cell mediated immune responses in humans. Because of this, it is important to know the similarities and the differences between E- and T-CD4 T cell population and to investigate the function of T- CD4 T cells during an immune response. Accordingly, the goal of the current application is to study T-CD4 T cells in depth. Aim 1 will determine to what extent they are different from or similar to E-CD4 T cells by employing several strategies including the DNA microarray assay. In Aim 2, we will study whether T-CD4 T cells mount an immune response in vivo similar to E-CD4 T cells. We will investigate whether T-CD4 T cells regulate the function of other immune cells and whether T-CD4 T cells can become memory cells. The last Aim will test the hypothesis that the presence of T-CD4 T cells regulates the development of atopic diseases. We will test this hypothesis by examining the role of T-CD4 T cells in the context of airway inflammation. The outcome of the proposed study will provide insights toward our understanding of T-CD4 T cells, which will help us to investigate T-CD4 T cells in immune diseases in human.